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Functional significance of thoracic vagal branches in the chicken
Respiration
Physiology
(1976)
27, 267-275
; North-Holland
Publishing
Company,
Amsterdam
FUNCTIONAL SIGNIFICANCE OF THORACIC VAGAL BRANCHES IN THE CHICKEN’
D. FRED Department Tex.
PETERSON
of Pharmacology,
78284,
and
U.S.
and THOMAS
The University
Department
of Texas Health
of Agriculture, Dela.
A.R.S., 19947.
E. NIGHTINGALE
Science Center at San Antonio,
San Antonio,
Poultry
Georgetown,
Research
Laboratory,
U.S.A.
Abstract. One minute electrical stimulation was used to excite the right and left cervical vagi as well as
specific points on, or branches of, the left thoracic vagus. Respiratory, heart rate and blood pressure responses were observed with the nerves intact and cut. Stimulation of either intact cervical vagus produced apnea, bradycardia and blood pressure depression. Stimulation of the cut ends after nerve section demonstrated that the heart rate and blood pressure effects were efferent and the respiratory change was afferent. No responses were observed due to stimulation of the vagus caudal to the lungs. Stimulation of cardiac branches reduced heart rate and blood pressure but did not produce significant respiratory effects. Middle and anterior pulmonary branches were found to contribute only to respiratory changes through afferent nerves. Sudden, sustained reduction of CO, in the airways produced immediate, sustained apnea. The data suggest that CO, sensitive thoracic receptors important in regulation of respiration are confined primarily to the lungs and that these receptors play no direct role in cardiovascular function. Blood pressure Carbon dioxide lung receptors Control of breathing
Heart frequency Vagus
Functional significance of specific afferent vagal fibers originating from branches in the thoracic cavity has not been well defined in birds. Nevertheless, the avian vagi are rich in both afferent and efferent fibers functional in respiratory and cardiovascular control (Fedde et al., 1963b; Johansen and Reite, 1964; Richards, 1969; Cohen and Schnall, 1970). Important information is carried from the lungs regarding regulation of respiration in response to changes in airway CO, (Peterson and Fedde, 1968). Receptors located in and near the heart have also been shown to be sensitive to CO, as well as to pressure and pH changes (Estavillo and Burger, 1973), but have not been given any physiological significance. Cervical section of the vagi produces a marked increase in respiratory amplitude accompanied by an extraordinary
Accepted
for publication
20 April
1976.
’ This work supported in part by AFOSR Grant #73-2525 267
268
D. F. PETERSON AND T. E. NIGHTINGALE
reduction in rate (King, 1966; Fedde et al., 1963a). Conflicting cardiovascular changes due to vagotomy have been reported (Johansen and Reite, 1964; Cohen and Schnall, 1970). In some species, thermal panting is abolished (Richards, 1968). Afferent cervical vagal stimulation has produced dramatic cardiovascular changes in some species (Johansen and Reite, 1964; King, 1966; Cohen and Schnall, 1970) while having little effect on others (Jones and Johansen, 1972). Weak stimuli produce a respiratory pattern similar to panting while strong stimuli cause apnea (Sinha, 1958 ; Richards, 1969). This study was undertaken to characterize respiratory and cardiovascular responses to supramaximal vagal stimulation in the chicken and to determine which thoracic vagal branches carry afferent or efferent information which mediates the responses seen during cervical vagal stimulation. The results have been compared to hypocapnia induced apnea.
Methods Twenty-two mature, male White Leghorns were anesthetized, i.m. with Equithesin (2.5 ml/kg, Jensen-Salsbery Labs). Each bird was placed on its back, the trachea isolated and ~nnulated, and the a~ominal air sacs ruptured through a midventral incision. Unidirectional respiration was then begun using warmed, humidified air (94 %) combined with CO, (6 %) at a total flow rate of 4 l/min. This combination produced normal spontaneous respiratory movements. (Avian anatomy and unidirectional artificial respiration have been detailed in a previous report; Fedde et al. 1969.) At this high gas-flow rate, abrupt,changes in ventilatory CO, produce abrupt changes in airway and, presumably, blood P,,, (Nightingale and Fedde, 1972). Body temperature was maintained at approximately 40 “C by a heating pad placed beneath the bird, and was monitored by a thermistor inserted into the rectum. Respiratory movements were measured with a strain gauge attached to the tip of the sternum (Fedde et al., 1963a). ECG and heart rate were recorded via needle electrodes placed in a standard Lead II ~on~guration. The pophteal artery was cannulated to measure blood pressure. All parameters were recorded on a Beckman, Type RM, Dynograph and Hewlett-Packard 3960 tape recorder. The thoracic cavity was opened by separation of the pectoral muscle from one side of the sternal plate and section of the coracoid bone at its sternal attachment. The left vagus nerve was exposed throughout the thorax and its branches identi~ed and separated from surrounding tissue. Each nerve to be stimulated was placed intact on bipolar platinum electrodes and stimulus parameters were chosen to maximize responses; 5-15 volts, l&20 pulses/set, 0.5-2.0 msec pulse duration, Order of nerve stimulation was chosen randomly and 4-5 minutes were allowed between periods of stimulation to assure that recorded parameters had returned to control levels. Nerves not being stimulated were covered with warm saline-soaked gauze to prevent drying. Unilateral and, subsequently, bilateral cervical vagotomies were performed
SIGNIFICANCE
OF THORACIC
VAGAL
269
BRANCHES
and stimuli applied to both ends of the cut nerves after each section. In several birds thoracic branches of the left vagus were cut to better separate afferent and efferent responses to stimulation of fibers to specific organs. Abrupt elimination of ventilatory CO, was accomplished by occluding the CO, gas line with a haemostat. CO, was readded by removal of the haemostat. Although this procedure slightly altered total flow, responses were not measureably different from those observed when CO, was simultaneously replaced by air (Peterson and Fedde, 1968). Significance was evaluated using a Student t-test. The determinations were made on either mean differences or comparisons of sample means. P < .05 was chosen as the accepted level of significance.
Results
When both vagi were intact, supramaximal stimulation of either vagus always produced immediate apnea, a dramatic reduction in heart rate and a fall in blood pressure (fig. 1; table 1). There were no significant differences in any of the responses to right or left vagal stimulation. CO2
200 1
REMOVAL
LEFT
VAGAL
STlMULATlON
Blood Pressure mmHg
Respiration
Fig.
I. Comparison
stimulation.
of responses
Stimulus
after CO, was readded
parameters
to CO,
removal
were: 10 V, 20/set,
to the ventilating immediate.
from the ventilating
gas and intact
2 msec pulse duration.
gas was delayed,
whereas
left cervical
after nerve stimulation,
The time bars equal one minute.
vagal
Note that onset of respiration recovery was
270
D. F. PETERSON AND T. E. NIGHTINGALE TABLE 1 Response to cervical vagal stimulation
Nerve stimulated
No.
Respiration /min
Mean heart rate /min % resting max
All nerves intact Left vagus (LV) Right vagus (RV)
19 8
Apnea Apnea
317-212 303-l 59
-33%” -48%*
87- 74 79- 61
- 15%* -23%;
10 3 8 3
Apnea NC Apnea NC
284282 346158 282-306 360-194
- 1% -54%** + 9%* -46%**
9& 94 103p 70 76111 109- 91
+ 4% -32%** +46%* - 17%**
Mean blood pressure mm Hg % resting max
Both vagi cut LV LV RV RV
central end peripheral end central end peripheral end
* P < .05. ** Inadequate sample for statistical analysis. NC = no change.
In order to determine which of the responses were direct efferent effects and which were reflex in nature, the vagi were cut and stimulations repeated. Neither unilateral nor bilateral vagotomy produced significant changes in resting heart rate or blood pressure. Respiration rate was significantly slowed due to unilateral vagotomy (24.414.1 cycles/min, P < .05)and further slowed due to bilateral vagotomy (14.1-10.1, P < .05). This was expected and has been well established in the literature both in spontaneously breathing (Johansen and Reite, 1964) and unidirectionally respirated birds of different species (Fedde et al., 1963a, b; Peterson and Fedde, 197 1). Stimulation of the central end of the cut left vagus always caused immediate apnea but no significant change in heart rate or blood pressure was demonstrated (table 1). This was true whether or not the right vagus was intact. Stimulation of the peripheral end of the left vagus had no effect on respiration although it caused a large fall in both heart rate and blood pressure (table 1). Again, these responses were observed whether or not the right vagus was intact. Stimulation of the central end of the cut right vagus produced a somewhat different effect than that observed due to stimulation of the left side. Apnea was always observed, while increases in both heart rate and blood pressure were observed whether or not the left vagus was intact (table 1). All of these changes were statistically significant (P < .0.5). Stimulation of the peripheral end of the right vagus did not elicit any change in respiration. A dramatic fall in heart rate and a definite fall in blood pressure were observed in three animals tested (table 1). Thus, in the chicken, only afferent fiber activity influences respiratory movements, whether the right or left vagus is being stimulated. Blood pressure and heart rate are both significantly reduced by activation of efferent vagal fibers while activation of afferent fibers in the right vagus can elicit increases in both heart rate and blood pressure.
SiGNr~CANCE
OF THORACIC
VAGAL
271
BRANCHES
In order to better define the origin of, or end-organ involved in the responses mediated by the vagus, the left thoracic vagus and its branches were located and stimulated. Sites of stimulation are indicated in fig. 2. No significant change was observed in any parameter measured when the intact whole vagus was stimulated near the diaphragm (AV) (table 2). Activation of intact cardiac branches (CB) caused a slight (8 %) increase in respiratory rate accompanied by a fall in respiratory amplitude (14 %), although 4 out of the 10 animals included in the averages showed no change. Both heart rate and blood pressure fell significantly (table 2). Responses observed in two animals after section of these cardiac branches indicated that direct efferent stimulation mirrored intact results for heart rate and blood pressure while no respiratory response occurred. Direct afferent stimulation in two animals caused no change in heart rate, blood pressure or respiratory rate. In one of these animals the amplitude of sternal movements was reduced by 9 % while the other animal was unaffected. heart
Fig. 2. Schematic drawing of the thoracic vagus and the branches stimulated. Areas stimulated include: AV = abdominal vagus; CB = cardiac branches, MPV = middle pulmonary vagus; AP = anterior pulmonary nerve; ATV = anterior thoracic vagus. Other abbreviations are: cb = carotid body; d = ‘diaphragm’ or pleural membrane; ng = nodoze ganglion; pt = parathyroid; t = thyroid; ub = ultimobranchial gland.
--_-
TABLE 2 Responses to intact vagal branch stimulation -___ AV n=6
CB n = 10
MPV n=8
AP n = 14
ATV n =i 14
CO, removal n = 22 -
NC NC
+23% -69%* -25%*
Apnea Apnea 0%
Apnea Apnea -31%*
Apnea Apnea
-5%
+3%
-1.5%
-1%
-Respiration
fate: amplitude:
Heart rate
-3%
+ 8% - 14% -27%+
Blood pressure ---
+2%
- 16%*
* P =z .OS. NC = no change. n = number of anjmals.
-.
-2%
272
D. F. PETERSON AND T. E. NIGHTINGALE
The posterior and middle pulmonary branches of the vagus are small and diffuse and, therefore, difficult to identify and stimulate as a unit. To investigate their influence, the middle pulmonary vagus (MPV) was stimulated near the anterior pole of the lung but below the anterior pulmonary nerve (fig. 2). In the face of a large fall in respiratory amplitude (69 %), respiratory rate increased 23 %. Heart rate fell significantly in this group of animals and average blood pressure fell but not significantly (table 2). In two animals, several branches of the middle pulmonary nerves were isolated and stimulated together. Apnea occurred but no changes were observed in heart rate or blood pressure. In most chickens the anterior pulmonary new (AP) is relatively large, discrete and easily isolated (fig. 2). Stimulation of the intact nerve caused immediate apnea (table 2). No significant change in heart rate or blood pressure was observed. The same responses were observed due to stimulation of the central end of the cut nerve in 3 animals, whereas no responses were seen due to stimulation of the peripheral end in two animals. Stimulation of the anterior thoracic vagus (ATV) above the pulmonary branches but below the carotid body produced apnea, a large, significant fall in heart rate and a fall in blood pressure (table 2). Surprisingly, 4 of 14 animals showed an increase in blood pressure. Consequently, the average fall in pressure did not prove to be significant. Apnea is easily produced in the chicken by a reduction in airway CO, (Peterson and Fedde, 1968). Typical responses to CO, change and left vagal stimulation are compared in fig. 1. All 22 animals were tested for their response to abrupt elimination of CO, from the ventilating gas. In every case, onset of apnea was observed to occur within one second but no significant changes were observed in heart rate or blood pressure (table 2). In general, whenever responses were observed due to either initiation or termination of nerve stimulation changes occurred immediately. This was always true of heart rate. Onset of apnea was always abrupt and similar to COz removal. On the other hand, recovery from apnea after nerve stimulation was always more rapid than after restoration of CO, (fig. 1).
Our results have quantified responses to supramaximal stimulation of the afferent and efferent vagi in regulation of respiration, heart rate and blood pressure in the chicken. In addition, we have identified the left thoracic vagal branches associated with each response. In this study, stimulation of the anterior pulmonary branch of the left vagus, or combined stimulation of middle and posterior pulmonary branches, produced rapid apnea, thus confirming the importance of pulmonary receptors. However, neural recordings have demonstrated receptors in the heart (Estavillo and Burger, 1973) which respond in similar fashion to CO, sensitive receptors in the avian lung
SIGNIFICANCE
OF THORACIC
VAGAL BRANCHES
273
(Fedde and Peterson, 1970). Furthermore, demonstration of CO1 sensitive receptors in the lungs functional in respiratory control (Peterson and Fedde, 1968) did not eliminate possible involvement of these cardiac receptors in respiratory control. Lack of a significant change in respiration during cardiac nerve stimulation in our study suggests that CO, sensitive cardiac receptors do not play an important role in regulation of respiration. The occurrence of a small reduction in extent of sternal movements associated with increased rate in 6 of 10 animals during stimulation, is puzzling. It may have been the result of slight stimulus current leak which affected the vagus. Submaximal vagal stimulation has previously been shown to increase respiratory rate while decreasing amplitude (Richards, 1969) and may activate fibers important in panting. Electrical stimulation of the cervical vagus which ordinarily induced apnea occasionally caused extremely low amplitude but rapid sternal movements. This was never observed during the respiratory response due to sudden elimination of CO, from the airways. Most likely, during nerve stimulation, fibers mediating apnea and fibers important in panting are both activated. At times, the apnea response apparently did not completely override the panting reflex. Our results demonstrated that the dominant cardiovascular responses to supramaximal activation of fibers in either vagus are direct efferent reductions in heart rate and blood pressure. It is probable that the fall in blood pressure was an indirect response to the abrupt, usually large fall in heart rate which, presumably, caused an immediate drop in cardiac output. Results of afferent stimulation after right vagotomy indicated a powerful pressor reflex. The reflex was present whether or not the left vagus was intact. A significant tachycardia was associated with central stimulation of the right vagus. Similar responses could not be elicited from the left vagus. These results are supported for right vagal stimulation by previous studies of Johansen and Reite (1964) in the duck and seagull. This apparent dissimilarity of function for afferent receptors, mediated by the two vagi, suggests a high degree of specificity for receptor location or afferent pathways. Such a specificity of pathway is not without some precident, since efferent influences on the heart have been shown to be highly pathway specific (Randall et al., 1972). Since the reflexly induced rise in avian blood pressure persisted after contralateral vagal section, it must be due to centrally mediated sympathetic tone (Jones and Johansen, 1972). The tachycardia produced in our experiments could not have accounted for the rise in blood pressure. It remains to be seen whether or not these two responses are related or are produced by separate mechanisms. In our experiments, significant changes in heart rate and blood pressure were not observed due to unilateral or bilateral vagal section. This is not consistent with experiments on other species of birds (Jones and Johansen, 1972). Double vagotomy in ducks and seagulls (Johansen and Reite, 1964) and pigeons (Cohen and Schnall, 1970) produces marked tachycardia, whereas in chickens, smaller but significant heart rate increases have been reported (Butler, 1967)‘. A more recent study found no change in heart rate in the chicken 15 minutes after bilateral cervical vagotomy
214
D. F. PETERSON
AND T. E. NIGHTINGALE
(Richards, 1969). Also, others have reported either increases (seagulls and ducks: Johansen and Reite, 1964) or decreases (pigeons : Cohen and Schnall, 1970) in blood pressure associated with bilateral vagotomy. The reason for the striking disparity in findings may be species related. On the other hand, bilateral vagotomy without artificial respiration presumably causes reduction in minute volume of respiratory gas which results in both hypoxia and hypercapnia. In older chickens it has been shown to lead to death within 3 hours (Fedde et al., 1963a). Previous studies have demonstrated that hypoxia in spontaneously breathing chickens causes tachycardia with no change in blood pressure (Butler, 1967). In unidirectionally ventilated chickens, heart rate and blood pressure both increased as respired CO, increased when 0, was constant (Ray and Feddy, 1969). When CO, was constant, blood pressure fell and heart rate rose as 0, was decreased in the ventilating gas. One may speculate that differences in responses to vagotomy may be explained by blood gas changes secondary to reduced minute ventilatory volume. Since stimulation of the anterior pulmonary nerve always produced apnea but had no effect on heart rate or blood pressure, the function of pulmonary receptors appear highly specific for respiratory regulation. On the other hand, stimulation of cardiac nerves had little influence on sternal movement and suggests that CO, sensitive receptors associated with fibers in these nerves are minimally involved in respiratory control.
Acknowledgements
The authors gratefully acknowledge the technical assistance of John Richardi. The authors are also grateful to Dr. H. 0. Stinnett for his critical review of the manuscript.
References Butler, P. J. (1967). The effect of progressive chicken. Cohen,
II. Electrical
hypoxia
on the respiratory
and cardiovascular
(1970). Medullary
cells of origin of vagal cardioinhibitory
of the dorsal motor
(1973). Avian cardiac
nucleus,
receptor
fibers in the
J. Camp. Neural. 140: 321-342.
activity
changes
by blood pressure,
carbon
and pH. Am. J. Physiol. 225: 1067-1071.
M. R., R. E. Burger
and R. L. Kitchell
(1963a).
The effect of anesthesia
following bilateral, cervical vagotomy in the fowl. Poulf. Sci. 42 Fedde, M. R., R. E. Burger and R. L. Kitchell (1963b). Localization
in respiratory
muscles
of Gallus
M. R. and D. F. Peterson
composition
in Gallus
domesticus.
J. Neurophysiol..
(1970). Intrapulmonary
domesticus.
receptor
and age on respiration
: 1212-l 223. of vagal afferents
maintenance of normal avian respiration. Poul~. Sci. 42 : 12241236. Fedde, M. R., P. D. DeWet and R. L. Kitchell (1969). Motor unit recruitment Fedde,
systems of the
191: 309-324.
stimulation
J. and R. E. Burger
dioxide, Fedde,
(Lend.)
D. H. and A. M. Schnall
pigeon. Estavillo,
J. Physic/o
pattern
involved
in the
and tonic activity
32: 995S1004. response
J. Physiol. (Lord.) 209: 609-625.
to changes
in airway-gas
SIGNIFICANCE OF THORACIC VAGAL BRANCHES
275
Johansen, K. and 0. B. Reite (1964). Cardiovascular responses to vagal stimulation and cardioaccelerator nerve blockade in birds. Comp. Biochem. Physiol. 12: 479487. Jones, D. R. and K. Johanserr(l972). The blood vascular system of birds. In: Avian Biology, Vol. 11, edited by D. S. Famer and J. R. King. New York, Academic Press. King, A. S. (1966). Afferent pathways in the vagus and their influence of avian breathing: A review. In: Physiology of the Domestic Fowl, edited by C. Horton-Smith and E. C. Amoroso. London, Oliver and Boyd, p. 302. Nightingale, T. E. and M. R. Fedde (1972). Determination of normal buffer line for chicken blood. Respir. Physiol. 14: 353-365. Peterson, D. F. and M. R. Fedde (1968). Receptors sensitive to carbon dioxide in lungs of chicken. Science 162: 1499-1501. Peterson, D. F. and M. R. Fedde (1971). Avian intrapulmonary CO,-sensitive receptors: a comparative study. Comp. Biochem. Physiol. 40: 425430. Randall, W. C., J. A. Armour, W. P. Geis and D. B. Lippincott (1972). Regional cardiac distribution of the sympathetic nerves. Fed. Proc. 31 : 1199-1208. Ray, P. J. and M. R. Fedde (1969). Responses to alterations in resporatory PO, and Pro* in the chicken. Respir. Physiol. 6: 135-143. Richards, S. A. (1968). Vagal control of thermal painting in mammals and birds. J. Physiol. (Land.) 199: 89-101.
Richards, S. A. (1969). Vagal function during respiration and the effects of vagotomy in the domestic fowl (Gallus domesticus). Comp. Biochem. Physiol. 29: 955-964.
Sinha, M. P. (1958). Vagal control of respiration as studied in the pigeon. Helu. Physiol. Acta 16: 58-72.
Physiology
(1976)
27, 267-275
; North-Holland
Publishing
Company,
Amsterdam
FUNCTIONAL SIGNIFICANCE OF THORACIC VAGAL BRANCHES IN THE CHICKEN’
D. FRED Department Tex.
PETERSON
of Pharmacology,
78284,
and
U.S.
and THOMAS
The University
Department
of Texas Health
of Agriculture, Dela.
A.R.S., 19947.
E. NIGHTINGALE
Science Center at San Antonio,
San Antonio,
Poultry
Georgetown,
Research
Laboratory,
U.S.A.
Abstract. One minute electrical stimulation was used to excite the right and left cervical vagi as well as
specific points on, or branches of, the left thoracic vagus. Respiratory, heart rate and blood pressure responses were observed with the nerves intact and cut. Stimulation of either intact cervical vagus produced apnea, bradycardia and blood pressure depression. Stimulation of the cut ends after nerve section demonstrated that the heart rate and blood pressure effects were efferent and the respiratory change was afferent. No responses were observed due to stimulation of the vagus caudal to the lungs. Stimulation of cardiac branches reduced heart rate and blood pressure but did not produce significant respiratory effects. Middle and anterior pulmonary branches were found to contribute only to respiratory changes through afferent nerves. Sudden, sustained reduction of CO, in the airways produced immediate, sustained apnea. The data suggest that CO, sensitive thoracic receptors important in regulation of respiration are confined primarily to the lungs and that these receptors play no direct role in cardiovascular function. Blood pressure Carbon dioxide lung receptors Control of breathing
Heart frequency Vagus
Functional significance of specific afferent vagal fibers originating from branches in the thoracic cavity has not been well defined in birds. Nevertheless, the avian vagi are rich in both afferent and efferent fibers functional in respiratory and cardiovascular control (Fedde et al., 1963b; Johansen and Reite, 1964; Richards, 1969; Cohen and Schnall, 1970). Important information is carried from the lungs regarding regulation of respiration in response to changes in airway CO, (Peterson and Fedde, 1968). Receptors located in and near the heart have also been shown to be sensitive to CO, as well as to pressure and pH changes (Estavillo and Burger, 1973), but have not been given any physiological significance. Cervical section of the vagi produces a marked increase in respiratory amplitude accompanied by an extraordinary
Accepted
for publication
20 April
1976.
’ This work supported in part by AFOSR Grant #73-2525 267
268
D. F. PETERSON AND T. E. NIGHTINGALE
reduction in rate (King, 1966; Fedde et al., 1963a). Conflicting cardiovascular changes due to vagotomy have been reported (Johansen and Reite, 1964; Cohen and Schnall, 1970). In some species, thermal panting is abolished (Richards, 1968). Afferent cervical vagal stimulation has produced dramatic cardiovascular changes in some species (Johansen and Reite, 1964; King, 1966; Cohen and Schnall, 1970) while having little effect on others (Jones and Johansen, 1972). Weak stimuli produce a respiratory pattern similar to panting while strong stimuli cause apnea (Sinha, 1958 ; Richards, 1969). This study was undertaken to characterize respiratory and cardiovascular responses to supramaximal vagal stimulation in the chicken and to determine which thoracic vagal branches carry afferent or efferent information which mediates the responses seen during cervical vagal stimulation. The results have been compared to hypocapnia induced apnea.
Methods Twenty-two mature, male White Leghorns were anesthetized, i.m. with Equithesin (2.5 ml/kg, Jensen-Salsbery Labs). Each bird was placed on its back, the trachea isolated and ~nnulated, and the a~ominal air sacs ruptured through a midventral incision. Unidirectional respiration was then begun using warmed, humidified air (94 %) combined with CO, (6 %) at a total flow rate of 4 l/min. This combination produced normal spontaneous respiratory movements. (Avian anatomy and unidirectional artificial respiration have been detailed in a previous report; Fedde et al. 1969.) At this high gas-flow rate, abrupt,changes in ventilatory CO, produce abrupt changes in airway and, presumably, blood P,,, (Nightingale and Fedde, 1972). Body temperature was maintained at approximately 40 “C by a heating pad placed beneath the bird, and was monitored by a thermistor inserted into the rectum. Respiratory movements were measured with a strain gauge attached to the tip of the sternum (Fedde et al., 1963a). ECG and heart rate were recorded via needle electrodes placed in a standard Lead II ~on~guration. The pophteal artery was cannulated to measure blood pressure. All parameters were recorded on a Beckman, Type RM, Dynograph and Hewlett-Packard 3960 tape recorder. The thoracic cavity was opened by separation of the pectoral muscle from one side of the sternal plate and section of the coracoid bone at its sternal attachment. The left vagus nerve was exposed throughout the thorax and its branches identi~ed and separated from surrounding tissue. Each nerve to be stimulated was placed intact on bipolar platinum electrodes and stimulus parameters were chosen to maximize responses; 5-15 volts, l&20 pulses/set, 0.5-2.0 msec pulse duration, Order of nerve stimulation was chosen randomly and 4-5 minutes were allowed between periods of stimulation to assure that recorded parameters had returned to control levels. Nerves not being stimulated were covered with warm saline-soaked gauze to prevent drying. Unilateral and, subsequently, bilateral cervical vagotomies were performed
SIGNIFICANCE
OF THORACIC
VAGAL
269
BRANCHES
and stimuli applied to both ends of the cut nerves after each section. In several birds thoracic branches of the left vagus were cut to better separate afferent and efferent responses to stimulation of fibers to specific organs. Abrupt elimination of ventilatory CO, was accomplished by occluding the CO, gas line with a haemostat. CO, was readded by removal of the haemostat. Although this procedure slightly altered total flow, responses were not measureably different from those observed when CO, was simultaneously replaced by air (Peterson and Fedde, 1968). Significance was evaluated using a Student t-test. The determinations were made on either mean differences or comparisons of sample means. P < .05 was chosen as the accepted level of significance.
Results
When both vagi were intact, supramaximal stimulation of either vagus always produced immediate apnea, a dramatic reduction in heart rate and a fall in blood pressure (fig. 1; table 1). There were no significant differences in any of the responses to right or left vagal stimulation. CO2
200 1
REMOVAL
LEFT
VAGAL
STlMULATlON
Blood Pressure mmHg
Respiration
Fig.
I. Comparison
stimulation.
of responses
Stimulus
after CO, was readded
parameters
to CO,
removal
were: 10 V, 20/set,
to the ventilating immediate.
from the ventilating
gas and intact
2 msec pulse duration.
gas was delayed,
whereas
left cervical
after nerve stimulation,
The time bars equal one minute.
vagal
Note that onset of respiration recovery was
270
D. F. PETERSON AND T. E. NIGHTINGALE TABLE 1 Response to cervical vagal stimulation
Nerve stimulated
No.
Respiration /min
Mean heart rate /min % resting max
All nerves intact Left vagus (LV) Right vagus (RV)
19 8
Apnea Apnea
317-212 303-l 59
-33%” -48%*
87- 74 79- 61
- 15%* -23%;
10 3 8 3
Apnea NC Apnea NC
284282 346158 282-306 360-194
- 1% -54%** + 9%* -46%**
9& 94 103p 70 76111 109- 91
+ 4% -32%** +46%* - 17%**
Mean blood pressure mm Hg % resting max
Both vagi cut LV LV RV RV
central end peripheral end central end peripheral end
* P < .05. ** Inadequate sample for statistical analysis. NC = no change.
In order to determine which of the responses were direct efferent effects and which were reflex in nature, the vagi were cut and stimulations repeated. Neither unilateral nor bilateral vagotomy produced significant changes in resting heart rate or blood pressure. Respiration rate was significantly slowed due to unilateral vagotomy (24.414.1 cycles/min, P < .05)and further slowed due to bilateral vagotomy (14.1-10.1, P < .05). This was expected and has been well established in the literature both in spontaneously breathing (Johansen and Reite, 1964) and unidirectionally respirated birds of different species (Fedde et al., 1963a, b; Peterson and Fedde, 197 1). Stimulation of the central end of the cut left vagus always caused immediate apnea but no significant change in heart rate or blood pressure was demonstrated (table 1). This was true whether or not the right vagus was intact. Stimulation of the peripheral end of the left vagus had no effect on respiration although it caused a large fall in both heart rate and blood pressure (table 1). Again, these responses were observed whether or not the right vagus was intact. Stimulation of the central end of the cut right vagus produced a somewhat different effect than that observed due to stimulation of the left side. Apnea was always observed, while increases in both heart rate and blood pressure were observed whether or not the left vagus was intact (table 1). All of these changes were statistically significant (P < .0.5). Stimulation of the peripheral end of the right vagus did not elicit any change in respiration. A dramatic fall in heart rate and a definite fall in blood pressure were observed in three animals tested (table 1). Thus, in the chicken, only afferent fiber activity influences respiratory movements, whether the right or left vagus is being stimulated. Blood pressure and heart rate are both significantly reduced by activation of efferent vagal fibers while activation of afferent fibers in the right vagus can elicit increases in both heart rate and blood pressure.
SiGNr~CANCE
OF THORACIC
VAGAL
271
BRANCHES
In order to better define the origin of, or end-organ involved in the responses mediated by the vagus, the left thoracic vagus and its branches were located and stimulated. Sites of stimulation are indicated in fig. 2. No significant change was observed in any parameter measured when the intact whole vagus was stimulated near the diaphragm (AV) (table 2). Activation of intact cardiac branches (CB) caused a slight (8 %) increase in respiratory rate accompanied by a fall in respiratory amplitude (14 %), although 4 out of the 10 animals included in the averages showed no change. Both heart rate and blood pressure fell significantly (table 2). Responses observed in two animals after section of these cardiac branches indicated that direct efferent stimulation mirrored intact results for heart rate and blood pressure while no respiratory response occurred. Direct afferent stimulation in two animals caused no change in heart rate, blood pressure or respiratory rate. In one of these animals the amplitude of sternal movements was reduced by 9 % while the other animal was unaffected. heart
Fig. 2. Schematic drawing of the thoracic vagus and the branches stimulated. Areas stimulated include: AV = abdominal vagus; CB = cardiac branches, MPV = middle pulmonary vagus; AP = anterior pulmonary nerve; ATV = anterior thoracic vagus. Other abbreviations are: cb = carotid body; d = ‘diaphragm’ or pleural membrane; ng = nodoze ganglion; pt = parathyroid; t = thyroid; ub = ultimobranchial gland.
--_-
TABLE 2 Responses to intact vagal branch stimulation -___ AV n=6
CB n = 10
MPV n=8
AP n = 14
ATV n =i 14
CO, removal n = 22 -
NC NC
+23% -69%* -25%*
Apnea Apnea 0%
Apnea Apnea -31%*
Apnea Apnea
-5%
+3%
-1.5%
-1%
-Respiration
fate: amplitude:
Heart rate
-3%
+ 8% - 14% -27%+
Blood pressure ---
+2%
- 16%*
* P =z .OS. NC = no change. n = number of anjmals.
-.
-2%
272
D. F. PETERSON AND T. E. NIGHTINGALE
The posterior and middle pulmonary branches of the vagus are small and diffuse and, therefore, difficult to identify and stimulate as a unit. To investigate their influence, the middle pulmonary vagus (MPV) was stimulated near the anterior pole of the lung but below the anterior pulmonary nerve (fig. 2). In the face of a large fall in respiratory amplitude (69 %), respiratory rate increased 23 %. Heart rate fell significantly in this group of animals and average blood pressure fell but not significantly (table 2). In two animals, several branches of the middle pulmonary nerves were isolated and stimulated together. Apnea occurred but no changes were observed in heart rate or blood pressure. In most chickens the anterior pulmonary new (AP) is relatively large, discrete and easily isolated (fig. 2). Stimulation of the intact nerve caused immediate apnea (table 2). No significant change in heart rate or blood pressure was observed. The same responses were observed due to stimulation of the central end of the cut nerve in 3 animals, whereas no responses were seen due to stimulation of the peripheral end in two animals. Stimulation of the anterior thoracic vagus (ATV) above the pulmonary branches but below the carotid body produced apnea, a large, significant fall in heart rate and a fall in blood pressure (table 2). Surprisingly, 4 of 14 animals showed an increase in blood pressure. Consequently, the average fall in pressure did not prove to be significant. Apnea is easily produced in the chicken by a reduction in airway CO, (Peterson and Fedde, 1968). Typical responses to CO, change and left vagal stimulation are compared in fig. 1. All 22 animals were tested for their response to abrupt elimination of CO, from the ventilating gas. In every case, onset of apnea was observed to occur within one second but no significant changes were observed in heart rate or blood pressure (table 2). In general, whenever responses were observed due to either initiation or termination of nerve stimulation changes occurred immediately. This was always true of heart rate. Onset of apnea was always abrupt and similar to COz removal. On the other hand, recovery from apnea after nerve stimulation was always more rapid than after restoration of CO, (fig. 1).
Our results have quantified responses to supramaximal stimulation of the afferent and efferent vagi in regulation of respiration, heart rate and blood pressure in the chicken. In addition, we have identified the left thoracic vagal branches associated with each response. In this study, stimulation of the anterior pulmonary branch of the left vagus, or combined stimulation of middle and posterior pulmonary branches, produced rapid apnea, thus confirming the importance of pulmonary receptors. However, neural recordings have demonstrated receptors in the heart (Estavillo and Burger, 1973) which respond in similar fashion to CO, sensitive receptors in the avian lung
SIGNIFICANCE
OF THORACIC
VAGAL BRANCHES
273
(Fedde and Peterson, 1970). Furthermore, demonstration of CO1 sensitive receptors in the lungs functional in respiratory control (Peterson and Fedde, 1968) did not eliminate possible involvement of these cardiac receptors in respiratory control. Lack of a significant change in respiration during cardiac nerve stimulation in our study suggests that CO, sensitive cardiac receptors do not play an important role in regulation of respiration. The occurrence of a small reduction in extent of sternal movements associated with increased rate in 6 of 10 animals during stimulation, is puzzling. It may have been the result of slight stimulus current leak which affected the vagus. Submaximal vagal stimulation has previously been shown to increase respiratory rate while decreasing amplitude (Richards, 1969) and may activate fibers important in panting. Electrical stimulation of the cervical vagus which ordinarily induced apnea occasionally caused extremely low amplitude but rapid sternal movements. This was never observed during the respiratory response due to sudden elimination of CO, from the airways. Most likely, during nerve stimulation, fibers mediating apnea and fibers important in panting are both activated. At times, the apnea response apparently did not completely override the panting reflex. Our results demonstrated that the dominant cardiovascular responses to supramaximal activation of fibers in either vagus are direct efferent reductions in heart rate and blood pressure. It is probable that the fall in blood pressure was an indirect response to the abrupt, usually large fall in heart rate which, presumably, caused an immediate drop in cardiac output. Results of afferent stimulation after right vagotomy indicated a powerful pressor reflex. The reflex was present whether or not the left vagus was intact. A significant tachycardia was associated with central stimulation of the right vagus. Similar responses could not be elicited from the left vagus. These results are supported for right vagal stimulation by previous studies of Johansen and Reite (1964) in the duck and seagull. This apparent dissimilarity of function for afferent receptors, mediated by the two vagi, suggests a high degree of specificity for receptor location or afferent pathways. Such a specificity of pathway is not without some precident, since efferent influences on the heart have been shown to be highly pathway specific (Randall et al., 1972). Since the reflexly induced rise in avian blood pressure persisted after contralateral vagal section, it must be due to centrally mediated sympathetic tone (Jones and Johansen, 1972). The tachycardia produced in our experiments could not have accounted for the rise in blood pressure. It remains to be seen whether or not these two responses are related or are produced by separate mechanisms. In our experiments, significant changes in heart rate and blood pressure were not observed due to unilateral or bilateral vagal section. This is not consistent with experiments on other species of birds (Jones and Johansen, 1972). Double vagotomy in ducks and seagulls (Johansen and Reite, 1964) and pigeons (Cohen and Schnall, 1970) produces marked tachycardia, whereas in chickens, smaller but significant heart rate increases have been reported (Butler, 1967)‘. A more recent study found no change in heart rate in the chicken 15 minutes after bilateral cervical vagotomy
214
D. F. PETERSON
AND T. E. NIGHTINGALE
(Richards, 1969). Also, others have reported either increases (seagulls and ducks: Johansen and Reite, 1964) or decreases (pigeons : Cohen and Schnall, 1970) in blood pressure associated with bilateral vagotomy. The reason for the striking disparity in findings may be species related. On the other hand, bilateral vagotomy without artificial respiration presumably causes reduction in minute volume of respiratory gas which results in both hypoxia and hypercapnia. In older chickens it has been shown to lead to death within 3 hours (Fedde et al., 1963a). Previous studies have demonstrated that hypoxia in spontaneously breathing chickens causes tachycardia with no change in blood pressure (Butler, 1967). In unidirectionally ventilated chickens, heart rate and blood pressure both increased as respired CO, increased when 0, was constant (Ray and Feddy, 1969). When CO, was constant, blood pressure fell and heart rate rose as 0, was decreased in the ventilating gas. One may speculate that differences in responses to vagotomy may be explained by blood gas changes secondary to reduced minute ventilatory volume. Since stimulation of the anterior pulmonary nerve always produced apnea but had no effect on heart rate or blood pressure, the function of pulmonary receptors appear highly specific for respiratory regulation. On the other hand, stimulation of cardiac nerves had little influence on sternal movement and suggests that CO, sensitive receptors associated with fibers in these nerves are minimally involved in respiratory control.
Acknowledgements
The authors gratefully acknowledge the technical assistance of John Richardi. The authors are also grateful to Dr. H. 0. Stinnett for his critical review of the manuscript.
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